Functional properties of amacrine cells in the mammalian retina

项目总结 该项目建议研究特定的无长突细胞(ACS)在视觉信号中的作用 哺乳动物的视网膜进行的处理。光诱发的电生理记录 活性将由ACS、神经节细胞(GC)和双极细胞完整地在体外完整地- 小鼠视网膜的贴片和切片准备保持在明视适应水平。这个 细胞的功能特性将通过投射到光感受器上的图像来探测 通过显微镜物镜。无长突细胞和神经节细胞的膜片钳记录 SOMAS将被用来测量刺激诱发的突触后电流，突触后 电势和尖峰反应。该项目的重点是阐明突触 3种基因标记的无长突细胞类型感受野特性的潜在机制。 目标1和目标2检查两种类型的无长突细胞的功能特性，即所谓的NOS-1 和NOS-2无长突细胞，通过其一氧化氮合酶的表达来识别 (否)。目的1将测试假设，即一氧化氮合酶-1ACS是关键的中间神经元控制 暗光水平下GC的环绕拮抗强度及其作用 通过GABA能突触连接到所有的急性冠脉综合征。我们将在两个NOS之间进行双重录音- 1个ACS和特定类型的GC，直接测试间接突触连接是否与 拟议中的电路。目标2将研究一氧化氮合酶-2ACS在赋予运动敏感性 小鼠体内特定类型的小视野GC。我们将使用ChR2的光遗传刺激 表达一氧化氮合酶以识别突触后靶点。突触后的目标将是 从形态和生理上确定来自NOS-2 ACS的输入将是 由配对录音证实。目标3侧重于一种新的无长突细胞类型，它是2AC之一 可以通过基因Gbx2的表达来识别的类型。我们将重点关注Gbx2+ 在内丛状层的亚层3(S3)中分层的ACS。S3-Gbx2+ACS高度 不同寻常，因为它们似乎不表达传统的抑制性或兴奋性 神经递质，表明它们代表了所谓的非GABA能的新种群， 非甘氨酸能(NGnG)型急性冠脉综合征。初步数据显示，这些nGnG AC是示踪剂偶联到 双极细胞。我们将量化这些nGnG S3-的时空感受场属性- Gbx2+ACS，并将测试他们通过双极突触产生输出的假设 细胞。为此，我们将对锥体双极细胞进行膜片钳记录。 S3-Gbx2+的光遗传刺激(ChR2表达)引起的去极化反应 ACES。总体而言，结果将揭示三个AC的功能属性和连通性 类型，并将决定它们在视网膜视觉处理中的角色。

PROJECT SUMMARY This project proposes to study the roles of specific amacrine cells (ACs) in the visual signal processing performed by the mammalian retina. Electrophysiological recordings of light evoked activity will be made from ACs, ganglion cells (GCs), and bipolar cells in intact in vitro whole- mount and slice preparations of mouse retina maintained at photopic adaptation levels. The functional properties of the cells will be probed using images projected onto the photoreceptors through the microscope objective. Patch-clamp recordings from amacrine and ganglion cell somas will be performed to measure the stimulus-evoked postsynaptic currents, postsynaptic potentials, and spiking responses. The project focusses on the elucidating the synaptic mechanisms underlying the receptive field properties of 3 genetically labelled amacrine cell types. Aims 1 and 2 examine the functional properties of two types of amacrine cells, so called NOS-1 and NOS-2 amacrine cells, which are identified by their expression of nitric-oxide synthase (NOS). Aim 1 will test the hypothesis that the NOS-1 ACs are key interneurons for controlling the strength of surround antagonism in GCs at scotopic light levels and that they exert their effects via GABAergic synaptic connections to AII ACs. We will make dual recordings between the NOS- 1 ACs and specific types of GCs, to directly test for indirect synaptic connections consistent with the proposed circuit. Aim 2 will examine the role of NOS-2 ACs in conferring motion-sensitivity to specific type of small-field GCs in the mouse. We will use optogenetic stimulation of ChR2 expressing NOS ACs to identify the postsynaptic targets. The postsynaptic targets will be identified morphologically and physiologically and inputs arising from NOS-2 ACs will be confirmed by paired recordings. Aim 3 focuses on a novel amacrine cell type that is one of 2 AC types that can be identified by their expression of the gene Gbx2. We will focus on the Gbx2+ ACs that stratify in sublamina 3 (S3) of the inner plexiform layer. The S3-Gbx2+ ACs are highly unusual because they appear to express none of the conventional inhibitory or excitatory neurotransmitters, indicating that they represent novel populations of so-called non-GABAergic, non-glycinergic (nGnG) ACs. Preliminary data show that these nGnG ACs are tracer coupled to bipolar cells. We will quantify the spatio-temporal receptive field properties of these nGnG S3- Gbx2+ ACs and will test the hypothesis that they make output via electrical synapses with bipolar cells. To do so, we will make patch-clamp recordings from cone bipolar cells in slice and measure depolarizing responses elicited by optogenetic stimulation (ChR2 expression) of the S3-Gbx2+ ACs. Overall, the results will reveal the functional properties and connectivity of the three AC types and will determine their roles in visual processing in the retina.